Histidine biosynthesis begins with condensation of ATP with phosphoribosyl pyrophosphate (PRPP) to form N1-(5′-phosphoribosyl)-ATP. Imidazole glycerol phosphate (IGP) synthase, a heterodimeric enzyme consisting of the hisF and hisH gene products, catalyzes the fifth step of histidine biosynthesis, wherein phosphoribulosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) and glutamine are transformed into glutamate, IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). This reaction is of the glutamine amidotransferase class. AICAR is a purine biosynthetic intermediate; thus there is a linkage between the purine and histidine biosynthetic pathways such that the purine ring removed in the first step of the histidine pathway is replenished by the couple between the reaction catalyzed by IGP synthase and the purine biosynthetic pathway.
It has been shown in a number of systems that missense mutations that decrease, but do not eliminate, the catalytic efficiency of the fourth step (formation of PRFAR from Pro-phoshporibosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide or 5′-ProFAR, catalyzed by 5′-ProFAR isomerase, the product of the hisA gene) or fifth step of histidine biosynthesis result in a biosynthetic limitation that is overcome by (a) histidine, (b) adenine or (c) a false feedback inhibitor of the first step the histidine pathway (Hartman et al. (1960) J. Gen. Microbiol. 22:323; Shedlovsky and Magasanik (1962) J. Biol. Chem. 237:3725; Shedlovsky and Magasanik (1962) J. Biol. Chem 237:3731; Galloway and Taylor (1980) J. Bacteriol. 144:1068; Shioi et al. (1982) J. Biol. Chem. 257:7969; Burton (1955) Biochem. J. 61:473; Burton (1957) Biochem. J. 66:488; Stougaard and Kennedy (1988) J. Bacteriol. 170:250). This result indicates that a high level flux through the partially blocked histidine biosynthetic pathway results in an ATP (energy) drain. Such blockage has been shown to have unique, deleterious pleiotropic effects upon a diversity of energy-intensive microbial processes including chemotaxis (Galloway and Taylor (1980) J. Bacteriol. 144:1068), DNA replication (Burton (1955) Biochem. J. 61:473; Burton (1957) Biochem. J. 66:488) and nitrogen fixation (Stougaard and Kennedy (1988) J. Bacteriol. 170:250). In each interrupted process, activity is restored by (a) histidine, (b) adenine or (c) a false feedback inhibitor of the first step in histidine biosynthesis.
These studies strongly suggest that enzymes encoded by the hisA, hisF or hisH genes will be useful for discovering herbicides and fungicides. The discovery of homologous biosynthetic pathways and corresponding enzymes in plants and fingi indicates that inhibition of such enzymes would be viable strategies for herbicidal control of unwanted vegetation and fungicidal control of plant disease. For example, inhibition of the fourth and fifth steps of histidine biosynthesis will result in the specific draining of the ATP pool to levels significantly lower than normal (Johnson and Taylor (1993) Applied Environ. Microbiol. 59:3509). This specific drain is achieved by having the histidine synthetic pathway operating at a high, near maximal, rate through the relief from allosteric feedback inhibition of the hisG-encoded enzyme, ATP phosphoribosyl transferase. By preventing the release of AICAR by the IGP synthase, the adenylate pool is drained. Although energy homeostasis can be maintained by simply re phosporylation of the adenylate to a high energy state, inhibition of the hisHF or hisA encoded enzymes traps the adenylate as histidine biosynthetic intermediates. Accordingly, lowered flux through the enzymes encoded by hisA and hisHF will cripple the cell's ability to carry out necessary metabolic processes.
Moreover, interruption of other steps in the histidine biosynthetic pathway in plants may also result in plant growth inhibition or death. For example, decrease or elimination of histidinol phosphate aminotransferase encoded by a plant homolog of hisC may inhibit conversion of glutamate to α-ketoglutarate and thereby have a detrimental effect on plant growth and development. The enzyme encoded by hisB is in part responsible for catalyzing the seventh and ninth steps of the histidine biosynthetic pathway. In the seventh step of the pathway D-erythro-1-(imidazol-4-yl)glycerol 3-phosphate is converted to 3-(imidazol-4-yl)-2oxopropyl phosphate by HisB. In the ninth step of the pathway histidinol phosphate is converted to histidinol by the action of HisB. Very little is know about HisB activity in plants, however, because this enzyme catalyzes two steps in the pathway. Interruption of HisB activity could severely alter normal histidine biosynthesis. Lastly, interruption of histidinol dehydrogenase activity (encoded by a homolog of the hisD gene), the enzyme that catalyzes the final step in the pathway, would prevent the formation of histidine. Finally, since the biosynthesis of histidine is energetically costly to the cell, inhibition of transformations at the later steps in the pathway would consume significant cellular energy resources without the formation of the expected end product, thus placing the affected cell at a disadvantage.
Thus, availability of the genes and their encoded enzymes has utility for herbicide and fungicide discovery via the design and implementation of cell-based screening and assay methodologies, enzyme-based screening and assay methodologies, rationale inhibitor design, x-ray crystallography, combinatorial chemistry and other modern biochemical and biotechnological methods.